The present invention relates to substances, their compositions, and methods for the regulation of cytokine activity, for instance, the up regulation or down regulation of Tumor Necrosis Factor alpha (TNF-xcex1) activity. In particular, substances and pharmaceutically acceptable compositions are disclosed which, when administered to a host in effective amounts, either inhibit or augment the secretion of active TNF-xcex1 by host cells. It is thought that the secretion of active cytokines, for example TNF-xcex1, by the host""s immune effector cells (e.g., the host""s activated macrophages) may be regulated by the methods of the present invention.
The present invention also relates to methods for the prevention and/or treatment of pathological processes or, conversely, the initiation of a beneficial immune system-related response involving the induction of cytokine production, secretion, and/or activity. Selected compositions of the present invention comprise an effective low dosage of a low molecular weight heparin (LMWH) to be administered at intervals of up to between five to eight days. Still other compositions include substances comprising carboxylated and/or sulphate oligosaccharides in substantially purified form obtained from a variety of primary sources including chromatographic separation and purification of LMWHs, enzymatically degraded heparin and enzymatically degraded extracellular matrix (DECM).
Individually, the substances, compositions containing same, and pharmaceutical compositions especially suited for parenteral, oral, or topical administration, inhibit or augment TNF-xcex1 secretion by resting T cells and/or macrophages in vitro in response to activation by immune effector cell activators, including, but not limited to, T cell-specific antigens, T cell mitogens, macrophage activators, residual extracellular matrix (RECM), fibronectin, laminin or the like. In vivo data, showing inhibition of experimental delayed type hypersensitivity (DTH), are also presented in further support of the in vitro results.
TNF-xcex1, a cytokine produced by monocytes (macrophages) and T lymphocytes, is a key element in the cascade of factors that produce the inflammatory response and has many pleiotropic effects as a major orchestrator of disease states (Beutler, B. and Cerami, A., Ann. Rev. Immunol. (1989) 7:625-655).
The biologic effects of TNF-xcex1 depend on its concentration and site of production: at low concentrations, TNF-xcex1 may produce desirable homeostatic and defense functions, but at high concentrations, systemically or in certain tissues, TNF-xcex1 can synergize with other cytokines, notably interleukin-1 (IL-1) to aggravate many inflammatory responses.
The following activities have been shown to be induced by TNF-xcex1 (together with IL-1); fever, slow-wave sleep, hemodynamic shock, increased production of acute phase proteins, decreased production of albumin, activation of vascular endothelial cells, increased expression of major histocompatibility complex (MHC) molecules, decreased lipoprotein lipase, decreased cytochrome P450, decreased plasma zinc and iron, fibroblast proliferation, increased synovial cell collagenase, increased cyclo-oxygenase activity, activation of T cells and B cells, and induction of secretion of the cytokines, TNF-xcex1 itself, IL-1, IL-6, and IL-8. Indeed, studies have shown that the physiological effects of these cytokines are interrelated (Philip, R: and Epstein, L. B., Nature (1986) 323(6083):86-89; Wallach., D. et al., J. Immunol. (1988) 140(9):2994-2999).
How TNF-xcex1 exerts its effects is not known in detail, but many of the effects are thought to be related to the ability of TNF-xcex1 to stimulate cells to produce prostaglandins and leukotrienes from arachidonic acid of the cell membrane.
TNF-xcex1, as a result of its pleiotropic effects, has been implicated in a variety of pathologic states in many different organs of the body. In blood vessels, TNF-xcex1 promotes hemorrhagic shock, down regulates endothelial cell thrombomodulin and enhances a procoagulant activity. It causes the adhesion of white blood cells and probably of platelets to the walls of blood vessels, and so, may promote processes leading to atherosclerosis, as well as to vasculitis.
TNF-xcex1 activates blood cells and causes the adhesion of neutrophils, eosinophils, monocytes/macrophages and T and B lymphocytes. By inducing IL-6 and IL-8, TNF-xcex1 augments the chemotaxis of inflammatory cells and their penetration into tissues. Thus, TNF-xcex1 has a role in the tissue damage of autoimmune diseases, allergies and graft rejection.
TNF-xcex1 has also been called cachectin because it modulates the metabolic activities of adipocytes and contributes to the wasting and cachexia accompanying cancer, chronic infections, chronic heart failure, and chronic inflammation. TNF-xcex1 may also have a role in anorexia nervosa by inhibiting appetite while enhancing wasting of fatty tissue.
TNF-xcex1 has metabolic effects on skeletal and cardiac muscle. It has also marked effects on the liver: it depresses albumin and cytochrome P450 metabolism and increases production of fibrinogen, l-acid glycoprotein and other acute phase proteins. It can also cause necrosis of the bowel.
In the central nervous system, TNF-xcex1 crosses the blood-brain barrier and induces fever, increased sleep and anorexia. Increased TNF-xcex1 concentration is associated with multiple sclerosis. It further causes adrenal hemorrhage and affects production of steroid hormones, enhances collagenase and PGE-2 in the skin, and causes the breakdown of bone and cartilage by activating osteoclasts.
In short, TNF-xcex1 is involved in the pathogenesis of many undesirable inflammatory conditions in autoimmune diseases, graft rejection, vasculitis and atherosclerosis. It may have roles in heart failure and in the response to cancer. For these reasons, ways have been sought to regulate the production, secretion, or availability of active forms of TNF-xcex1 as a means to control a variety of diseases.
The prime function of the immune system is to protect the individual against infection by foreign invaders such as microorganisms. It may, however, also attack the individual""s own tissues leading to pathologic states known as autoimmune diseases. The aggressive reactions of an individual""s immune system against tissues from other individuals are the reasons behind the unwanted rejections of transplanted organs. Hyper-reactivity of the system against foreign substances causes allergy giving symptoms like asthma, rhinitis and eczema.
The cells mastering these reactions are the lymphocytes, primarily the activated T lymphocytes, and the pathologic inflammatory response they direct depends on their ability to traffic through blood vessel walls to and from their target tissue. Thus, reducing the ability of lymphocytes to adhere to and penetrate through the walls of blood vessels may prevent autoimmune attack, graft rejection and allergy. This would represent a new therapeutic principle likely to result in better efficacy and reduced adverse reactions compared to the therapies used today.
Atherosclerosis and vasculitis are chronic and acute examples of pathological vessel inflammation. Atherosclerosis involves thickening and rigidity of the intima of the arteries leading to coronary diseases, myocardial infarction, cerebral infarction and peripheral vascular diseases, and represents a major cause of morbidity and mortality in the Western world. Pathologically, atherosclerosis develops slowly and chronically as a lesion caused by fatty and calcareous deposits. The proliferation of fibrous tissues leads ultimately to an acute condition producing sudden occlusion of the lumen of the blood vessel.
TNF-xcex1 has been shown to facilitate and augment human immunodeficiency virus (HIV) replication in vitro (Matsuyama, T. et al., J. Virol. (1989) 63(6):2504-2509; Michihiko, S. et al., Lancet (1989) 1(8648):1206-1207) and to stimulate HIV-1 gene expression, thus, probably triggering the development of clinical AIDS in individuals latently infected with HIV-1 (Okamoto, T. et al., AIDS Res. Hum. Retroviruses (1989) 5(2):131-138).
Hence, TNF-xcex1, like the inflammatory response of which it is a part, is a mixed blessing. Perhaps in understanding its physiologic function, one may better understand the purpose of inflammation as a whole and gain insight into the circumstances under which xe2x80x9cTNF-xcex1 deficiencyxe2x80x9d and xe2x80x9cTNF-xcex1 excessxe2x80x9d obtain. How best to design a rational and specific therapeutic approach to diseases that involve the production of this hormone may thus be closer at hand.
Heparin is a glycosaminoglycan, a polyanionic sulfated polysaccharide, which is used clinically to prevent blood clotting as an antithrombotic agent. In animal models, heparin has been shown to reduce the ability of autoimmune T lymphocytes to reach their target organ (Lider, O. et al., Eur. J. Immunol. (1990) 20:493-499). Heparin was also shown to suppress experimental autoimmune diseases in rats and to prolong the allograft survival in a model of skin transplantation in mice, when used in low doses (5 xcexcg for mice and 20 xcexcg for rats) injected once a day (Lider, O. et al., J. Clin. Invest. (1989) 83:752-756).
The mechanisms behind the observed effects are thought to involve inhibition of release by T lymphocytes of enzyme(s) necessary for penetration of the vessel wall, primarily the enzyme heparanase that specifically attacks the glydosaminoglycan moiety of the sub-endothelial extracellular matrix (ECM) that lines blood vessels (Naparstek, Y. et al., Nature (1984) 310:241-243). Expression of the heparanase enzyme is associated with the ability of autoimmune T lymphocytes to penetrate blood vessel walls and to attack the brain in the model disease experimental autoimmune encephalomyelitis (EAE).
European Patent Application EP 0114589 (Folkman et al.) describes a composition for inhibition of angiogenesis in mammals in which the active agents consist essentially of (1) heparin or a heparin fragment which is a hexasaccharide or larger and (2) cortisone or hydrocortisone or the 11-xcex1 isomer of hydrocortisone. According to the disclosure, heparin by itself or cortisone by itself are ineffective; only the combination of both gives the desired effects. Although there is no proof in the literature that there is a connection between angiogenesis and autoimmune diseases, the description on page 5 of the patent application connects angiogenesis with psoriasis and with arthritis, indicating the use of high doses of 25,000 units to 47,000 units of heparin per day (i.e., about 160 to about 310 mg per day).
Horvath, J. E. et al., in Aust. N.Z.J. Med. (1975) 5(6):537-539, describe the effect of subanticoagulant doses of subcutaneous heparin on early renal allograft function. The daily dosage is high (5000 U or about 33 mg) and the conclusion of the study is that heparin in subanticoagulant doses has no effect on early graft function or graft survival and that it may be associated with increased hemorrhagic complications.
Toivanen, M. L. et al., Meth. and Find. Exp. Clin. Pharmacol. (1982) 4(6):359-363, examined the effect of heparin in high dosage (1000 U/rat or about 7 mg/rat) in the inhibition of adjuvant arthritis in rats and found that heparin enhanced the severity of the rat adjuvant arthritis.
PCT Patent: Application PCT/AU88/00017 published under No. WO88/05301 (Parish et al.) describes sulphated polysaccharides that block or inhibit endoglycosylase activity, such as heparanase activity, for use as antimetastatic and anti-inflammatory agents. Heparin and heparin derivatives, such as periodate oxidized, reduced heparins, that had negligible anticoagulant activity, were shown to have antimetastatic and anti-inflammatory activity when used in dosages within, the range of 1.6-6.6 mg per rat daily, administered by constant infusion (corresponding to 75-308 mg daily for an adult human patient).
Heparin and heparan sulfate are closely related glycosaminoglycan macromolecules. The degradation products of these polymeric macromolecules, which are termed low molecular weight heparins (LMWH), may have the same or greater pharmacologic effects on the blood clotting system as the parent macromolecules. Furthermore, because there is extensive but incomplete post-synthetic processing of the polymer""s basic disaccharide subunit, glucuronic acid and N-acetyl glucosamine, the LMWH will be a heterogeneous mixture not only of sizes but also of chemical compositions (See Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 8th Ed., (Pergamon Press, New York, 1990) pp. 1313-1315. Methods to obtain low molecular weight products from heparin, which are useful as anticoagulants, are described in the art. These methods seek to optimize the persistence in vivo or the extent of hemorrhagic side effects of their products (See, for example, Alpino R. R., et al., U.S. Pat. No. 5,010,063; Choay, J., et al., U.S. Pat. No. 4,990,502; Lopez, L. L., et al., U.S. Pat. No. 4,981,955). Others teach the use of affinity chromatographic methods to obtain low molecular weight products (See, for example, Rosenberg, R. D., et al., U.S. Pat. No. 4,539,398 and Jordan, R. E., et al., U.S. Pat. No. 4,446,314).
Psuja, P., as reported in Folio Haematol. (Leipz), (1987) 114:429-436, studied the effect of the heterogeneity of heparins on their interactions with cell surfaces. Psuja reported that there are moderate affinity receptors for LMWH (Dd=5.6 xcexcM) found on cultured endothelial cells, but he determined that the upper limit of the fraction of LMWH bound to these receptors was less than 1% of total LMWH.
Other workers have demonstrated effects of LMWH on the metabolism of a variety of cultured cell types. Asselot-Chapel, C., et al., in Biochem. Pharmacol. (1989) 38:895-899 and Biochem. Biophys. Acta, (1989) 993:240-244, report that LMWH cause cultured smooth muscle cells to decrease the ratio of type III to type I collagen and fibronectin synthesis. Rappaport, R. in U.S. Pat. No. 4,889,808, teaches that LMWH can cause human diploid pulmonary fibroblasts, cultured in the absence of serum, to respond to LMWH by increased secretion of tissue plasminogen activator and related proteins.
Effects of LMWH on complex multicellular systems have been reported. The work of Folkman et al. and Lider et al., in EPO Application 0114589 and J. Clin. Invest. (1989) 83:752:756, have been noted above. In addition, Diferrante, N., in published International Application WO 90/03791, teaches the use of LMWH to inhibit the reproduction of HIV in cultures of C8166 transformed human lymphocytes (ALL). However, none of the prior art experiments that have studied the effects of LMWH on cellular metabolism has considered that the heterogeneity of LMWH may produce antagonistic effects. Furthermore, none has shown or suggested a regulatory effect on cytokine activity based on the use of substantially pure oligosaccharide substances.
In the present invention, substances are disclosed which are capable of regulating cytokine activity in a mammalian subject and which are comprised of a carboxylated and/or sulfated oligosaccharide in a substantially purified form. In particular, the substance exhibits a consistent: (a) inhibitory xe2x80x9cRxe2x80x9d value of about 200,000%xc3x97(xcexcg/gm)xe2x88x921 or more as determined from an in vivo bioassay that measures the relative inhibition of experimental DTH reactions in mice that have been treated with varying dosages of said substance ranging from 0 to about 2 xcexcg/gm mouse; or (b) augmentative xe2x80x9cRxe2x80x9d value of about 0.03%xc3x97(pg/ml)xe2x88x921 or more as determined from an in vitro bioassay that measures the relative activity of TNF-xcex1 that is secreted by activated human CD4+ T cells in the presence of varying concentrations of said substance from 0 to about 1xc3x97107 pg/ml. Preferred substances exhibit in vivo inhibitory xe2x80x9cRxe2x80x9d values selected from the group consisting of 300,000, 400,000, 500,000 and 600,000%xc3x97(xcexcg/gm)xe2x88x921 or more.
Furthermore, the substances of the present invention having an inhibitory effect on the secretion of active TNF-xcex1 may, in addition, exhibit a consistent inhibitory xe2x80x9cRxe2x80x9d value of at least about 0.4%xc3x97(pg/ml)xe2x88x921 as determined from an in vitro bioassay that measures the relative activity of TNF-xcex1 that is secreted by activated human CD4+T cells in the presence of varying concentrations of said substance from 0 to about 1xc3x97107 pg/ml.
In one embodiment of the present invention, the carbohydrate or oligosaccharide has a molecular weight of no more than about 3000 daltons, preferably lying in the range of about 400 to about 2000 daltons, most preferably between about 400 and about 1100 daltons. Generally, substances of the present invention which inhibit TNF-xcex1 activity, as determined by biological assays (described more fully, below) comprise molecules of various sugar units of which the basic unit of activity is associated with a disaccharide. However, larger oligosaccharide chains of up to about 10 sugar units, containing the basic disaccharide unit of activity can also function to inhibit TNF-xcex1 activity. On the other hand, the substances of the present invention, which act to augment the observed activity of TNF-xcex1, are generally of two types: (i) relatively higher molecular weight aggregates of low molecular weight molecules that, in a non-aggregated state, show inhibitory activity; and (ii) disaccharide or monosaccharide subunits that have lost sulfate groups (i.e., have experienced at least some desulfation).
When purified these substances or the compositions that contain them are substantially free of other substances that exert the opposite or antagonistic effect. Thus, a substance exhibiting inhibitory activity (xe2x80x9cdownxe2x80x9d regulation) in a substantially purified form would be substantially free not only of other substances, in general, but of other substances that exhibit augmentation or retard the inhibitory activity of the xe2x80x9cdownxe2x80x9d regulator. The situation would, of course, be reversed in the case of an augmentative substance (i.e., xe2x80x9cupxe2x80x9d regulators), in which the substance would be substantially free of other substances, particularly those that xe2x80x9cdownxe2x80x9d regulate or antagonize augmentation.
The phrase xe2x80x9cregulatory effectxe2x80x9d includes both the up regulation or down regulation of any process affecting the availability or resulting activity in vivo or in vitro of cytokines, in general, including IL-1, IL-6, IL-8 and, in particular, TNF-xcex1. Thus, compositions of the-present invention may exert a regulatory effect on the host production of TNF-xcex1, on the host secretion of TNF-xcex1, on the extracellular availability of TNF-xcex1, or on the active forms of TNF-xcex1 in a host. For instance, but not wishing to be limited by theory, the instant invention may act to elicit the secretion of a substance, such as a protein, which may bind to TNF-xcex1, change its conformation, and, consequently, affect its biological activity. It is also possible that the compositions of the present invention may, in penetrating activated T cells or macrophages, bind to particular oligonucleotide sequences and, thus, affect transcriptional or translational processes that ultimately alter protein synthesis. The compositions may also work through binding to cell surface receptors.
To simplify the following discussion, reference will be made, among others, to the xe2x80x9csecretion of active TNF-xcex1xe2x80x9d or the regulation of the xe2x80x9cactivity of TNF-xcex1xe2x80x9d with the understanding that a much broader meaning is to be attached to these phrases which encompasses the actual mechanism that is responsible for or the actual manner by which the observed augmentation or inhibition of TNF-xcex1 activity is effected by the substances and compositions of the present invention.
The substances of the present invention comprise a carboxylated and/or sulfated oligosaccharide moiety that may be obtained from natural sources, including living organisms. For example, active substances have been isolated and purified from low molecular weight heparin (LMWH) fractions, as well as extracellular matrices that have been degraded by the action of an enzyme, e.g., heparanase derived from animals (mammals) or microorganisms (bacteria). Yet another source of active substances is enzyme-treated heparin (e.g., endoglycosylase-degraded heparin).
Hence, the term xe2x80x9csubstantially purified formxe2x80x9d means that specific steps have been taken to remove non-active components, or components that have an opposing effect, from the oligosaccharide substances and to isolate the active moiety or moieties from mixtures or supernatants, such as those obtained from enzymatic degradation. Specifically, the substances claimed in the present invention are obtained from a rigorous chromatographic process, in which low pressure size-exclusion gel chromatography (i.e., chromatography on Sephadex columns) is but an initial step in the purification scheme. Subsequent to the low pressure separation, high pressure liquid chromatographic (HPLC) techniques are used to isolate individual component oligbsaccharides. Preferably, these steps have resulted in the purification of the individual active substances to substantial homogeneity.
Such a preferred purification step may include, for example, passing mixtures containing the active substance (e.g., fractions obtained from low pressure gel chromatography) through gel permeation HPLC or strong anion exchange (SAX) HPLC columns. Thus, substances comprising oligosaccharides selected from the group consisting of di-, tri-, tetra-, penta-, or hexasaccharides, preferably disaccharides, have been observed and isolated. The oligosaccharides of the present invention are carboxylated and/or sulfated and are, therefore, negatively charged. Particular embodiments of the invention preferentially include disaccharides having three negatively charged groups. Those that exhibit a specific inhibitory activity possess a molecular weight ranging from about 400 to about 2000, preferably, about 400 to about 1100.
The present invention also provides a bioassay for quantifying the effect of a test substance on the secretion of active TNF-xcex1. The bioassay comprises the steps of preincubating human CD4+ T cells in a medium with varying concentrations of a test substance, adding a constant amount of an activator effective to elicit the secretion of TNF-xcex1 by the T cells in the absence of said test substance, collecting the medium after a sufficient period of time, and subsequently testing the activity of the TNF-xcex1 in the medium. Preferably, the human CD4+ T cells are obtained from peripheral blood mononuclear leukocytes (PBL). Suitable immune effector cell activators include, but are not limited to, T cell-specific antigens, mitogens, macrophage activators, residual extracellular matrix (RECM, defined in Section 4, below), laminin, fibronectin, and the like.
The present invention relies on the specific regulatory activity of particular substances as determined by in vitro and in vivo bioassays described in greater detail, below. Briefly, the substances useful in the present invention display a regulatory (either inhibitory or augmentative) activity relating to the induction of the secretion of active TNF-xcex1 which is dose dependent. That is, a plot of the percent inhibition or augmentation versus the dose (e.g., pg/ml of substance) gives rise to a bell-shaped curve from which a maximum percent inhibition (Inhmax) or augmentation (Augmax) is readily apparent. Thus, for every point on such a plot, a xe2x80x9cratioxe2x80x9d between the percent inhibition or augmentation and the concentration or dose can be calculated. In the present case, a xe2x80x9cspecific regulatory activityxe2x80x9d or xe2x80x9cRxe2x80x9d value can be obtained from the ratio of the maximum percent inhibition or augmentation (i.e., Inhmax or Augmax) and the concentration or dose of test substance which gave rise to such maximum percent regulatory value. Furthermore, an xe2x80x9cRxe2x80x9d value can be obtained for each bioassay. Hence, an xe2x80x9cRxe2x80x9d value can be associated from an in vitro mouse spleen cell assay, an ex vivo mouse spleen assay, an in vitro human PBL assay, and an in vivo assay based on experimental DTH reaction. If no effect is observed, an xe2x80x9cRxe2x80x9d value of zero is assigned.
Another object of the present invention is a method of regulating cytokine activity in a mammalian subject comprising administering to said subject an amount of a substance effective to inhibit or augment the activity of a cytokine in said subject, said substance comprising a carboxylated and/or sulfated oligosaccharide in a substantially purified form and said substance exhibiting a consistent: (a) non-zero inhibitory xe2x80x9cRxe2x80x9d value as determined from (i) an in vitro bioassay that measures the relative activity of TNF-xcex1 that is secreted by activated human CD4+ T cells in the presence of varying concentrations of said substance from 0 to about 1xc3x97107 pg/ml, and/or (ii) an in vivo bioassay that measures the relative inhibition of experimental DTH reaction in mice that have been treated with varying dosages of said substance ranging from 0 to about 2 xcexcg/gm mouse; or (b) non-zero augmentative xe2x80x9cRxe2x80x9d value as determined from an in vitro bioassay that measures the relative activity of TNF-xcex1 that is secreted by activated human CD4+ T cells in the presence of varying concentrations of said substance from 0 to about 1xc3x97107 pg/ml.
Yet another object of the present invention is a method of using the active substance for the preparation of a pharmaceutical preparation useful for the treatment of the host, which method comprises combining the substance with a pharmaceutically acceptable carrier to provide a unit dose, preferably of low dosage, having an effective amount of the substance. The pharmaceutical preparation may also comprise a stabilizing agent, for example, protamine, in an amount sufficient to preserve a significant, if not substantial, proportion of the initial activity of the substance over an extended period, e.g., about 100 percent over about 3 days. At storage temperatures below room temperature, e.g., about xe2x88x9210 to about 10xc2x0 C., preferably 4xc2x0 C., more of the initial activity is preserved, for up to about 4 months.
Because the pharmaceutical compositions of the present invention are contemplated for administration into humans, the pharmaceutical compositions are preferably sterile. Sterilization is accomplished by any means well known to those having ordinary skill in the art, including use of sterile ingredients, heat sterilization or passage of the composition through a sterile filter.
It should also be evident that a primary object of the present invention is to provide a method of treating a host, such as a mammalian subject, suffering from a medical condition the severity of which can be affected by the activity of a cytokine in the host comprising administering to such host an active substance comprising the oligosaccharides of the instant invention in substantially purified form or the pharmaceutical compositions that can be prepared from same. Depending on the medical condition of the particular host, substances or compositions can be administered which either reduce the availability or activity of TNF-xcex1 or, conversely, enhance TNF-xcex1 induction or amplify its activity. Such compositions or pharmaceutical preparations may be administered at low dosage levels and at intervals of up to about 5-8 days, preferably, once a week. Pharmaceutical compositions containing oligosaccharide (e.g., mono-, di-, tri-, or tetrasaccharides, preferably, comprising a disaccharide) substances for parenteral, oral, or topical administration may be administered daily according to convenience and effectiveness and at dosages that would be readily determined by routine experimentation by one of ordinary skill.
The present invention is also related to pharmaceutical preparations for the prevention and/or treatment of pathological processes involving the induction of active TNF-xcex1 secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH) present in a low effective dose for administration at intervals of up to about 5-8 days and which LMWH is capable of inhibiting in vitro secretion of active TNF-xcex1 by resting T cells and/or macrophages in response to T cell-specific antigens, mitogens, macrophage activators, residual extracellular matrix (RECM), laminin, fibronectin, and the like.
In a particular embodiment of the present invention the LMWH of the pharmaceutical preparation has an average molecular weight of from about 3,000 to about 6,000 and, furthermore, may be administered every fifth or seventh day.
It is also an objective of the present invention to provide a pharmaceutical preparation to be administered at intervals of up to about 5-8 days for the prevention and/or treatment of pathological processes involving the induction of active TNF-xcex1 secretion comprising a pharmaceutically acceptable carrier and a low molecular weight heparin (LMWH) present in a low effective dose.
Active substances and compositions of the present invention are capable of inhibiting experimental delayed type hypersensitivity (DTH) reactions to an applied antigen as evidenced by a reduction in the induration observed after the application of the antigen to the skin up to about five to seven days after the administration of the substance or pharmaceutical composition of same relative to the induration observed after the application of the antigen to the skin in the absence of or after recovery from the administration of the substance or pharmaceutical composition of same. Examples of the applied antigen include, but are not limited to, tetanus, myelin basic protein, purified protein derivative, oxazolone, and the like.
Furthermore, it is an objective of the present invention to provide compositions or pharmaceutical preparations that may be administered in any manner as dictated by the particular application at hand including, but not limited to, enteral administration (including oral or rectal) or parenteral administration (including topical or inhalation with the aid of aerosols). In preferred embodiments, the pharmaceutical compositions of the present invention are administered orally, subcutaneously, intramuscularly, intraperitoneally or intravenously.
Thus, the present invention is useful, for example, in delaying or preventing allograft rejection and treating or preventing a variety of pathological processes such as those related to autoimmune diseases, allergy, inflammatory diseases (in particular, inflammatory bowel disease), or acquired immunodeficiency syndrome (AIDS). The present invention also finds utility in the treatment of diabetes type I, periodontal disease, skin diseases, liver diseases, uveitis, rheumatic diseases (in particular, rheumatoid arthritis), atherosclerosis, vasculitis, or multiple sclerosis.
Moreover, the present invention is useful, in the treatment of tumors, viral infections and bacterial infections by administering a substance of the invention so as to augment the""secretion of active TNF-xcex1. Examples of tumor treatment include, but are not limited to, the treatment of breast, colon and prostate cancers as well as lymphomas and other basal cell carcinomas. Bacterial infection treatments include, but are not limited to, the treatment of diphtheria, streptococcus, pneumonia, gonorrhea, leprosy, and tuberculosis. Similarly, examples of viral infections which can be treated by the invention include, but are not limited to, the treatment of influenza, hepatitis, gastroenteritis, mononucleosis, bronchiolitis, and meningitis.
In particular pharmaceutical compositions of the present invention, low effective doses of the prescribed LMWH active substance are present. Typically, the pharmaceutical composition contains a single low dose unit of less than 5 mg of LMWH active substance, preferably from about 0.3 to about 3 mg, and most preferably contains a single low dose unit of from 1 to 1.5 mg.
The present invention also contemplates broadly a method of using a low molecular weight heparin (LMWH) which is capable of inhibiting in vitro secretion of active TNF-xcex1 by resting T cells and/or macrophages in response to immune effector cell activators for the preparation of a pharmaceutical preparation to be administered at intervals of up to about 5-8 days for the prevention and/or treatment of pathological processes involving induction of TNF-xcex1 secretion which method comprises combining a low effective dose of the LMWH with a pharmaceutically acceptable carrier.
Yet another object of the present invention relates to methods for providing sources of active substances according to the present invention which comprise fractionating low molecular weight heparins, enzymatically degrading intact heparin (DH), or enzymatically degrading extracellular matrix (DECM).
A still further object of the present invention is to provide a method of treating a subject or host suffering from a pathological process involving induction of active TNF-xcex1 secretion comprising administering to such subject or host a pharmaceutical composition, as described above, at intervals of up to about 5-8 days, preferably once a week. As further described above, pharmaceutical compositions comprising active oligosaccharide may also be administered daily or up to weekly intervals.
The present invention also provides a pharmaceutical composition for the inhibition of the production of active TNF-xcex1 comprising a disaccharide of the formula (I) or its pharmaceutically acceptable salt 
in which X1 is hydrogen or sulfate; X2 is hydrogen or sulfate; and X3 is sulfate or acetyl, provided that if X3 is sulfate, then at least one of X1 or X2 is sulfate and if X3 is acetyl, then both X1 and X2 are sulfates; and a pharmaceutically acceptable carrier. In particular, the pharmaceutical composition may comprise a disaccharide which is 2-O-sulfate-4-deoxy-4-en-iduronic acid-(xcex1-1,4)-2-deoxy-2-Nxe2x80x94
Yet another aspect of the present invention relates to a pharmaceutical composition for augmenting the production of active TNF-xcex1 comprising a non-sulfated N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
Also contemplated by the present invention is a method of inhibiting the production of an active cytokine in a subject comprising administering to the subject, for example, a mammal, such as a human patient, an effective amount of a disaccharide of the formula (I) or its pharmaceutically acceptable salt 
in which X1 is hydrogen or sulfate; X2 is hydrogen or sulfate; and X3 is sulfate or acetyl, provided that if X3 is sulfate, then at least one of X1 or X2 is sulfate and if X3 is acetyl, then both X1 and X2 are sulfates. Another method relates to augmenting the production of an active cytokine in a subject comprising administering to the subject an effective amount of a disaccharide which is 4-deoxy-4-en-iduronic acid-(xcex1-1,4)-2-deoxy-2-N-acetylglucosamine or a pharmaceutically acceptable salt thereof. Consistent with the objectives of the present invention, such methods include the daily or, preferably, weekly sulfateglucosamine, 4-deoxy-4-en-iduronic acid-(xcex1-1,4)-2-deoxy-2-N-sulfate-6-O-sulfateglucosamine, 2-O-sulfate-4-deoxy-4-en-iduronic acid-(xcex1-1,4)-2-deoxy-2-N-sulfate-6-O-sulfateglucosamine, or 2-O-sulfate-4-deoxy-4-en-iduronic acid-(xcex1-1,4)-2-deoxy-2-N-acetyl-6-O-sulfateglucosamine.
The present invention also contemplates a pharmaceutical composition for augmenting the production of active TNF-xcex1 comprising 4-deoxy-4-en-iduronic acid-(xcex1-1,4)-2-deoxy-2-N-acetylglucosamine or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such pharmaceutical compositions may, of course, be adapted for various routes of administration including, but not limited to, parenteral administration, oral administration, or topical administration.
Furthermore, a pharmaceutical composition is provided for the inhibition of the production of active TNF-xcex1 comprising a compound which is an N-sulfated or N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof. Such compound, if N-sulfated, has at least one other sulfate group and, if N-acetylated, has at least two sulfate groups. It should be noted that because of the unsaturation (i.e., the double bond at C-4 to C-5) at the xe2x80x9curonicxe2x80x9d acid portion of certain of the disaccharides of interest, there is no stereochemistry associated with the C-6 carboxyl group that is essentially in the plane of the six-membered ring. Hence, when the double bond at C-4 to C-5 is present, an iduronic acid is the same as a glucuronic acid. Consequently, the term xe2x80x9curonicxe2x80x9d acid is meant to encompass either a glucuronic or an iduronic acid. Likewise, a xe2x80x9curonoxe2x80x9d group can mean either an idurono or glucurono group. administration of the respective compounds or their pharmaceutically acceptable salts.
The above-mentioned methods may also be utilized for inhibiting or augmenting the production of an active cytokine in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of the present invention.
The present invention also contemplates a method of using a compound which is an N-sulfated or N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof, the compound if N-sulfated having at least one other sulfate group and the compound if N-acetylated having at least two sulfate groups for the preparation of a pharmaceutical composition for the prevention or treatment of a medical condition caused by or related to the inappropriate production of TNF-xcex1.
Also contemplated is a method of using a compound which is a non-sulfated N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition for the treatment of a medical condition responsive to an increased production of TNF-xcex1.
Likewise, methods of preventing or treating a medical condition caused by or related to the inappropriate production of an active cytokine in a subject are also provided comprising administering to the subject an effective amount of a compound which is an N-sulfated or N-acetylated 4-deoxy-4-en-glucoronoglucosamine or a pharmaceutically acceptable salt thereof, the compound if N-sulfated having at least on other sulfate group and the compound if N-acetylated having at least two sulfate groups. Methods of treating a medical condition responsive to an increased production of an active cytokine in a subject area also provided which comprise administering to the subject an effective amount of a compound which is a non-sulfated N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof. Such treatments are particularly useful in cases involving an autoimmune disease, a neoplastic condition or some form of infection, including those induced by viral, bacterial or fungal agents.
Other objects of the present invention concern methods of protecting a subject from the harmful effects of exposure to radiation comprising administering to the subject an effective amount of a compound which is an N-sulfated or N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof, the compound if N-sulfated having at least one other sulfate group and the compound if N-acetylated have at least two sulfate groups. Typically, the compounds of the present invention are administered to the subject prior to radiation exposure. Most advantageously, the radioprotective properties of the disclosed compounds may be exploited during radiation therapy.
Further, methods of suppressing allograft rejection in a subject are contemplated comprising administering to the subject an effective amount of a compound which is an N-sulfated or N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof, the compound if N-sulfated having at least one other sulfate group and the compound if N-acetylated have at least two sulfate groups. The allograft may, of course, include an organ transplant, including, but not limited to, heart, liver, kidney or bone marrow transplants. The disclosed methods may also apply to skin grafts.
Yet another object relates to a method of suppressing the expression of an adhesion molecule in a subject comprising administering to the subject an effective amount of a compound which is an N-sulfated or N-acetylated 4-deoxy-4-en-glucuronoglucosamine or a pharmaceutically acceptable salt thereof, the compound if N-sulfated having at least one other sulfate group and the compound if N-acetylated have at least two sulfate groups. Examples of such adhesion molecules include, but are not limited to, ICAM-1 or ELAM-1.
Also disclosed is an in vitro bioassay for quantifying the effect of a test substance on the secretion of active TNF-xcex1 comprising preincubating human CD4+ T cells in a medium with varying concentrations of a test substance, adding a constant amount of an activator effective to elicit the secretion of TNF-xcex1 by the T cells in the absence of the test substance, collecting the medium after a sufficient period of time, and subsequently testing the activity of the TNF-xcex1 in the medium.
Further objects of the present invention will become apparent to those skilled in the art upon further review of the following disclosure, including the detailed descriptions of specific embodiments of the invention.